Workers in the plant sciences have developed techniques for removing the rigid, cellulose wall of plant cells in order to obtain wall-less cells commonly known as protoplasts. Plant protoplasts have potential economic value in that they permit plant scientists to apply microbiological techniques for the production of improved plant varieties significant in agriculture. Thus, by taking advantage of naturally occurring variation or variation induced artifically by chemicals or radiation in the protoplasts, plant scientists may be able to select out plants bearing agriculturally useful new traits. The significant new advantage presented by plant protoplasts is that each single celled protoplast is theoretically capable of giving rise to a whole plant and subsequently to a new type of crop. Consequently, by the application of standard microbiological techniques, plant scientists may be able to deal with vastly more plants than ever before possible and therefore to detect much more readily the very rare events which lead to useful new varieties.
In addition plant protoplasts have considerable potential value in the production of hybrids, i.e. offspring from two different plant varieties, species or genera, which have important commercial value in agriculture. Protoplasts of different plant species may be fused to form a hybrid protoplast which is potentially capable of growing into a hybrid plant even in instances where the hybrid may be unobtainable because of genetic barriers using conventional plant breeding techniques.
Plant protoplasts are expected to be the point of attack for manipulation of the plant genome using recombinant DNA techniques as a way of improving plant varieties. The lack of a cell wall barrier offers significant advantages for the introduction of genetically engineered DNA.
Most of the predicted advantages of using plant cell protoplasts in agricultural improvements have not been realized in practice because of the difficulty in culturing and/or regenerating the cultured cells into intact plants. With the technique currently available it has been possible to achieve regeneration of protoplasts into whole plants in only a very limited number of instances and with marginal efficiencies. Regeneration from fused protoplasts extracted from plant leaves and stems has proven to be a viable method for the production of hybrids in a few instances, e.g. tobacco plant protoplasts have responded to known regeneration techniques and a number of hybrids have been thus produced. U.S. Pat. No. 3,832,801 shows a process for the regeneration of hybrids of the parent species Nicotiana glauca and N. langsdorffi.
Most of the important crop plants are refractory to currently available regeneration technology. For example, with respect to tomato plants, no process has been found for the regeneration of a viable plant from protoplasts.
The tomato (Lycopersicon esculentum) is an important crop plant and has proven to be one of the most versatile of cultivated plants. The agronomic value of the tomato was improved through the transfer of disease-resistance characteristics from other Lycopersicon species. Sexual hybrids between L. esculentum and the more closely related species are easily obtained. However, interspecific incompatibilities with some species limits the value of sexual hybridization for the introduction of important traits from wild species. Cell fusion and genetic engineering methods have been proposed to facilitate gene transfer in the tomato, but these methods require efficient plant regeneration from cultured protoplasts.
F. J. Zapata, P. K. Evans, J. B. Power and E. C. Cocking, "The effect of temperature on the division of leaf protoplasts of Lycopersicon esculentum and Lycopersicon peruvianum," Plant Sci. Lett. 8:119-124 (1977) disclose research with regard to the effect of temperature on the initiation and maintenance of cell division and callus formation with regard to mesophyll protoplasts of two tomato species, and note that while cell wall regeneration has been performed with isolated protoplasts, no sustained cell division has been reported when these protoplasts were cultured.
W. K. Coleman and R. I. Greyson, "Promotion of root initiation by giberrellic acid in leaf discs of tomato Lycopersicon esculentum cultured in vitro," New Phytol. 78:47-54 (1977) detail the use of gibberellic acid as a growth-promoting hormone.
A. C. Cassells and M. Barlass, "A Method for the isolation of stable mesophyll protoplasts from tomato leaves throughout the year under standard conditions," Physiol. Plant. 42:236-242 (1978) postulate that the failure of existing methods to regenerate plants from tomato mesophyll protoplasts is due to deficiencies in the culture medium, and suggest growing the protoplast-donating plants under specified conditions of nutrition and lighting.
E. Pojnar, J. H. M. Willison and E. C. Cocking, "Cell-wall regeneration by isolated tomato-fruit protoplasts," Protoplasma 64:460-480 (1969) have determined that cell wall regeneration may be helpful in the survival of isolated tomato protoplasts and disclose a method for such cell wall regeneration.
It is noted that none of these references detail a successful method for the regeneration of a cultivated tomato plant from protoplasts, and rather describe the extensive research in the art which has been directed to attaining such a result.
V. Padmanabhan, E. F. Paddock and W. R. Sharp, in "Plantlet formation from Lycopersicon esculentum leaf callus," Can. J. Bot. 52:1429-1432 (1974) describe the micropropagation of plants from cultivated tomato leaf callus, i.e. from callused leaf sections wherein the protoplasts are retained within their cell walls. This technique produces clones of a plant for varying purposes but does not enable the production of hybrids or allow genetic modification of the plants due to the fact that the protoplasts within the leaf sections are not freed for such manipulation.
Additional publications suggest the usefulness of a method for the production, from isolated protoplasts, of mature plants which have heretofore been incapable of such regeneration. For example, M. L. Kalil and A. C. Hildebrandt, "Effects of Agrobacterium sp. on cell cultures of tomato," Phyton 28:177-186 (1971) describe the isolation of protoplasts and the use of cell culturing methods to access aspects of the tumorization process. B. E. Ellis, "Non-differential sensitivity to the herbicide metribuzin in tomato cell suspension cultures," Can. J. Plant Sci. 58:775-778 (1978) describes the isolation of protoplasts and the use of cell suspension cultures to test the differential tolerance of tomato plants to the herbicide metribuzin.
C. P. Meredith, in "Response of cultured tomato cells to aluminum," Plant Sci. Lett. 12:17-24 (1978) describes the isolation of tomato protoplasts to determine significant differences in aluminum tolerance in cultivated plants. This reference discloses the measuring of such response by weighing individual callus pieces before and after exposure to aluminum in the culture medium. In a similar manner, C. P. Meredith, "Selection and characterization of aluminum-resistant variants from tomato cultures," Plant Sci. Lett. 12:25-34 (1978) describes the selection of callused cultured resistant cells, again tested by weight gain of the callus in a cultured medium. It is noted, at page 26, that the variant cells cannot be induced to regenerate plants.
A. C. Cassells, "Uptake of charged lipid vesicles by isolated tomato protoplasts," Nature 275:760 (1978) describes a method for the insertion of DNA into protoplasts which would be useful if plants could be regenerated therefrom.
While methods for the extraction of protoplasts which are capable of cell fusion or genetic manipulation are known, and workers have heretofore been successful in promoting the division of such cells, I am unaware of any process which enables the formation of viable, callused cell colonies, i.e. colonies which will eventually form mini-calli and shoots, from the isolated protoplasts of plants such as the cultivated tomato, cotton, members of the cucurbitaceae family, the brassica species, the euphorbia species, legumes such as soybeans and other plants which have hetertofore resisted such regeneration. This has appeared to be a critical step in the regeneration of tomato plants, as the mini-calli turn brown and die even in the most successful of the prior attempts at regeneration. As used herein, the term "mini-calli" refers to the callus which is formed on plant cell colonies, the mini-calli being barely visible, i.e. about two to three millimeters in diameter.
Thus, it has been a desideratum in the plant sciences to provide a reliable procedure for the production of viable callused cell colonies and for plant regeneration from protoplasts in plant species such as the cultivated tomato which have heretofore failed to respond to plant regeneration techniques.
In accordance with the present invention, a process is provided for plant regeneration from the protoplasts of plants which have heretofore resisted such techniques, and is described in some detail with regard to plant regeneration from protoplasts of the cultivated tomato.
While I do not wish to be bound by any particular theory, it is thought that certain plants which fail to regenerate from protoplasts do so due to the plant cell's inability to survive the cell extraction and culturing procedures. Specifically, cells which have developed in a natural state fail to grow in vitro because their natural hormone levels are incompatible with those of the artificial media. The natural hormones have been evolved by nature to produce a plant, not a protoplast which is viable in vitro. Consequently, the protoplasts fail to form viable calli, which then develop to form shoots, roots and plants. Thus, a method is provided whereby the source of the protoplast to be regenerated, i.e. the proto plast-donating plant, is a plant which has been preconditioned and treated, from its seed form throughout its life until the harvesting of the protoplasts, to prepare the cells for the regeneration regimen.
A seed consists of a dormant embryo, together with a quantity of stored nutrients and hormones, and one or two integuments which differentiate into the protective seed coat. The embryo may be an undifferentiated mass of cells, e.g. in the orchid family, but is usually more highly organized and consists of a short axis which is called the hypocotyl, which at one end bears a primitive root called the radicle and at the other end a terminal bud or plumule. Borne laterally at the apex of the hypocotyl just below the plumule are one or more seed leaves or cotyledons, which are filled with stored food material and form the greater part of the seed. The hormones which regulate the development of the embryo, the germination of the seed and the growth of the plant originate from within the embryonic tissue and are thus regarded as endogenous in origin. The major portion of these endogenous hormones is produced by the radicle.
Germination is the development of the embryo into a young plant. It becomes completed when the young plant is independent of the food and hormones stored in the seed. In most seeds, the first visible sign of germination is a swelling of the seed, which is a result of an increased water content. Often the seed coats are ruptured by the swelling of the contents of the seed. Respiration increases greatly, and much energy is made available to those regions where active growth occurs, that is, the hypocotyl and the plumule, which often push out of the seed to form a shoot. Finally, the radicle pushes out of the seed and attaches itself, by means of root hairs, to the soil particles. The roots then begin absorbing water and nutrients from the soil. Eventually, the plumule elongates and the first true leaves of the plant appear. With their formation, the plant becomes independent of the seed.
According to the invention, the protoplast is isolated from a protoplast-donating organism which has been grown in the substantial absence of endogenous hormones, as opposed to isolating the protoplast from a natural plant. The growth of the donating organism in the substantial absence of endogenous material is thought to precondition the organism so that the protoplasts are prepared for the in vitro environment and artificial hormone levels required by the regeneration procedure. For example, the protoplast-donating plants may be preconditioned by being grown from seed-generated shoots which have had the radicle neutralized or removed so that the nutrients and hormones contained in those portions do not substantially contribute to the growth of the shoot.
In another aspect of the invention, the protoplasts are cultured in a defined media sequence having a controlled regimen with regard to osmoticum, ammonia-producing salts and hormones to support and regulate the further growth of the protoplast and the formation of colonies, calli, roots and young plants.
Other varied preconditioning steps may be desirable for the protoplast regeneration of a particular plant. For example, it may be particularly helpful to grow the protoplast-donating plants in an artificial medium which is substantially free of all growth regulating hormones in order to enhance the extraction of viable protoplasts therefrom. Maintaining the donating plants in the absence of light for a period prior to the protoplast extraction has been shown to aid in the extraction of the protoplasts therefrom. However, it is thought essential to the practice of the invention that the protoplast-donating organism be deprived of a substantial portion of its normal endogenous growth regulators to ensure the survival and viable growth of the isolated protoplasts.
With regard to the growth media of the present invention, the terms "growth medium" or "nutrient medium" as used herein comprise a solution of the mineral salts and vitamins necessary for plant growth, generally containing combined nitrogen, potassium, phosphorous, calcium, sulphur and magnesium with traces of iron, boron, zinc, copper and various organic substances. Thus, the growth media employed in the various steps of the regeneration process may be any medium understood by those skilled in the art to provide the nutrients necessary for plant cell growth. However, it should be noted that improvements in the survival and growth of the protoplasts result from providing for a substantial similarity among the nutrient media and solutions employed in the described process so that the transport shock to the protoplasts, cell colonies, calli, shoots and roots is reduced. As used herein, the term "substantially similar" is intended to mean that aside from constituents which must be increased or decreased at a particular point in order to have a specific effect on growth or survival, such as sugars, ammonium salts, buffers and various hormones or vitamins, the components of the medium or solution do not essentially vary beyond the limits expressed herein.
Specifically, with regard to the media that support the isolated protoplasts and colonies formed therefrom, i.e. the free cell media, it is essential that the osmoticum (a measure of the osmotic pressure of the fluid quantified as the molarity of the sugars therein) should be increased to provide support for the wall-less cells, and the ammonium and nitrogen-containing salts which produce ammonia must be reduced or substantially eliminated. In the free cell media as well as in other media and solutions, specific hormones are included to promote the particular effect. Specific examples of the effect of various hormones and ammonium ions on callus formation are set forth in "Callus Formation from Leaf Mesophyll Protoplasts of Three Lycopersicon Species", Zapata et al., Plant Science Letters, 23 (1981) 41-46, which is hereby incorporated by reference.
While the invention is hereinafter described with regard to tomato cultivar Red Cherry and VF-36 varieties, UC-82, Lorz Cherry, Floradade, Cocktail Cherry, Manapal and VFNT-Cherry varieties have also been regenerated from protoplasts according to the method of the invention. Other crop plants which have heretofore been difficult or impossible to regenerate from somatic cells such as cotton, legumes, e.g. soybeans, members of the cucurbitaceae family, the brassica species and the euphorbia species are also generated according to the process hereinafter set forth, and the method of the invention provides distinct advantages in the protoplast regeneration of all plants and improves regeneration processes heretofore known.